Gravitropism in cut flower stalks of snapdragon.

The negative gravitropic response of cut flower stalks is a complex multistep process that requires the participation of various cellular components acting in succession or in parallel. The process was particularly characterized in snapdragon (Antirrhinum majus L.) spikes with regard to (1) gravity stimulus perception associated with amyloplast reorientation; (2) stimulus transduction mediated through differential changes in the level, action and related genes of auxin and ethylene and their possible interaction; (3) stimulus response associated with differential growth leading to stalk curvature; (4) involvement of cytosolic calcium and actin cytoskeleton. Results show that the gravity-induced amyloplast reorientation, differential over-expression of two early auxin responsive genes and asymmetrical distribution of free IAA are early events in the bending process. These precede the asymmetrical ethylene production and differential stem growth, which was derived from initial shrinkage of the upper stem side and a subsequent elongation of the lower stem side. Results obtained with various calcium- and cytoskeleton-related agents indicate that cytosolic calcium and actin filaments may play essential roles in gravitropism-related processes of cut flower stalks. Therefore, modulators of these two physiological mediators may serve as means for controlling any undesired gravitropic bending.

Feasibility study of repeated harvesting of menthol from biologically viable Mentha x piperata using ultrasonic extraction.

To potentially replace the conventional destructive extraction process, we have shown the feasibility of devising a novel technique that uses ultrasound to nonlethally and repeatedly extract menthol from biologically viable peppermint plants (Mentha x piperita). Our results show that plants ultrasonicated for 1 h at 22 degrees C in a standard 40 kHz ultrasonic bath could release approximately 17.8 microg of menthol per gram of leaf tissue (2% of total product). The amount of menthol release increases with the time of treatment and is greatly affected by the temperature of the ultrasonic bath water. An increase from 2% to 12% of total product was observed when the temperature was increased from 22 degrees C to 39 degrees C. When the temperature effects were isolated, the mechanism of the product release was found to be that of cavitation. The treated plants remained viable and were ready for the subsequent ultrasound extraction after approximately 4 days of recuperation. However, the amount of product released is reduced in subsequent extractions. Scanning electron micrographs indicate that there are two mechanisms involved in extraction: (1) the diffusion of product through the cuticle of peppermint glandular trichomes and (2) the exudation of the product from broken and damaged trichomes. This study has shown the possibility of using an on-line ultrasonic, nondestructive extraction method to continuously release intracellular plant metabolites from the plants while maintaining the plant's viability.

Changes in starch content in oat (Avena sativa) shoot pulvini during the gravitropic response.

In order to determine if components of the signal transduction pathway are involved in starch metabolism during the gravitropic response, the effects of inhibitors of phosphoprotein phosphatases and protein kinases (OA), and calcium channel blockers (LaCl3), on gravitropic bending and starch levels in gravisensitive node/pulvini of oat shoots were examined. Among the compounds tested, okadaic acid (OA) and lanthanum chloride (LaCl3) showed the strongest inhibitory effects on the negative gravitropic curvature response in oat shoot node/pulvini. At the same time, they caused a rapid loss of starch in graviresponding pulvini based on a quantitative analysis of starch levels in the bending tissues over 48 h periods. These two compounds act initially to block the net increase in starch content that occurs during the early stages (0-9 h) in graviresponding oat shoot pulvini. As a result, starch levels drop precipitously in shoots treated with OA and LaCl3, starting at time zero of gravistimulation by reorientation. These findings suggest that protein dephosphorylation and calcium play a role in starch metabolism in oat shoot pulvini in response to a gravistimulation signal. They also indicate that the amount of starch present in the chloroplast gravisensors in oat shoot pulvini may determine the rate of upward bending in graviresponding pulvini.

Permeabilization of metabolites from biologically viable soybeans (Glycine max).

Chemical permeabilization has been widely studied for the release useful metabolites from many types of plant cells and tissues. In this study, the effect of 0-30% (v/v) of aqueous methanol solutions were used to permeabilize soybeans for the release of two isoflavonoids: daidzein and genistein. The release of these metabolites increases with increasing methanol concentrations. The amounts of daidzein and genistein released can increase up to 40- and 86-fold, respectively, when incubated in a 30% (v/v) methanol solution for 24 h compared with those incubated with water only. The effect of methanol on the release rates is primarily due to an increase in solubility of the stored daidzein and genistein (14- to 18-fold) inside the seeds, thus maximizing the concentration gradients for metabolite release. However, the viability of the seeds dropped with increase in methanol concentrations and the incubation time. The viability of soybeans (indicated by their ability to germinate) after permeabilization treatment with 0-20% (v/v) methanol solutions was maintained above 80% throughout the 24 h, whereas no seeds were found to be viable when 30% (v/v) methanol solution was used. The permeability coefficients (P) of daidzein and genistein were found to increase as the methanol concentration used was increased. These P values were estimated to range from 1.1 x 10(-)(9) to 1.9 x 10(-)(8) m/s and 1.0 x 10(-)(9) to 1.7 x 10(-)(8) m/s, respectively. The increase in P can be attributed primarily to an increase in the partition coefficient of the metabolites in the soybean seedcoats. An empirical correlation is proposed in which the log P values are described as a function of the metabolite molecular weights and the partition coefficients of the metabolites between octanol and water, K(oct/water), which was modified to include the effect of methanol present. Knowledge obtained from this study will help provide useful selection criteria for chemical permeabilization of plant tissues, such as seeds, with minimal loss in their viability.

A role for inositol 1,4,5-trisphosphate in gravitropic signaling and the retention of cold-perceived gravistimulation of oat shoot pulvini.

Plants sense positional changes relative to the gravity vector. To date, the signaling processes by which the perception of a gravistimulus is linked to the initiation of differential growth are poorly defined. We have investigated the role of inositol 1,4,5-trisphosphate (InsP(3)) in the gravitropic response of oat (Avena sativa) shoot pulvini. Within 15 s of gravistimulation, InsP(3) levels increased 3-fold over vertical controls in upper and lower pulvinus halves and fluctuated in both pulvinus halves over the first minutes. Between 10 and 30 min of gravistimulation, InsP(3) levels in the lower pulvinus half increased 3-fold over the upper. Changes in InsP(3) were confined to the pulvinus and were not detected in internodal tissue, highlighting the importance of the pulvinus for both graviperception and response. Inhibition of phospholipase C blocked the long-term increase in InsP(3), and reduced gravitropic bending by 65%. Short-term changes in InsP(3) were unimpaired by the inhibitor. Gravitropic bending of oat plants is inhibited at 4 degrees C; however, the plants retain the information of a positional change and respond at room temperature. Both short- and long-term changes in InsP(3) were present at 4 degrees C. We propose a role for InsP(3) in the establishment of tissue polarity during the gravitropic response of oat pulvini. InsP(3) may be involved in the retention of cold-perceived gravistimulation by providing positional information in the pulvini prior to the redistribution of auxin.

Effects of staurosporine, okadaic acid and sodium fluoride on protein phosphorylation in graviresponding oat shoot pulvini.

This study focuses on the characterization of protein phosphorylation in the gravitropic response in oat shoot pulvini through the use of inhibitors of this process, namely staurosporine, okadaic acid and sodium fluoride. These three inhibitors reduce gravitropic curvature and cause changes in the phosphorylation of 38- and 50-kDa soluble proteins which show different levels of phosphorylation between lower and upper halves of gravistimulated pulvini. A kinetic analysis of phosphorylation shows that the 38- and 50-kDa soluble proteins exhibit different levels of phosphorylation between lower and upper halves of graviresponsive pulvini at 5 min after initiation of gravistimulation of stems. In addition, the phosphorylation of 63- and 70-kDa proteins from a total membrane preparation increases in lower halves of the pulvini following gravistimulation. These phosphoproteins are not found in the plasma membrane fraction. Taken together, at least four kinds of phosphoproteins are gravi-related. Of these, the 38- and 50-kDa soluble phosphoproteins may be involved in the regulation of early stages of the gravitropic response.

Inhibition of the gravitropic response of snapdragon spikes by the calcium-channel blocker lanthanum chloride.

The putative Ca(2+)-channel blocker LaCl3 prevented the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes (S. Philosoph-Hadas, S. Meir, I. Rosenberger, A.H. Halevy [1996] Plant Physiol 110: 301-310) and inhibited stem curvature to a greater extent than vertical and horizontal stem elongation at the bending zone. This might indicate that LaCl3, which modulates cytosolic Ca2+, does not influence general stem-growth processes but may specifically affect other gravity-associated processes occurring at the stem-bending zone. Two such specific gravity-dependent events were found to occur in the bending zone of snapdragon spikes: sedimentation of starch-containing chloroplasts at the bottom of stem cortex cells, as seen in cross-sections, and establishment of an ethylene gradient across the stem. Our results show that the lateral sedimentation of chloroplasts associated with gravity sensing was prevented in cross-sections taken from the bending zone of LaCl3-treated and subsequently gravistimulated spikes and that LaCl3 completely prevented the gravity-induced, asymmetric ethylene production established across the stem-bending zone. These data indicate that LaCl3 inhibits stem curvature of snapdragon spikes by preventing several gravity-dependent processes. Therefore, we propose that the gravitropic response of shoots could be mediated through a Ca(2+)-dependent pathway involving modulation of cytosolic Ca2+ at various stages.

A comparative survey of leguminous plants as sources of the isoflavones, genistein and daidzein: implications for human nutrition and health.

Over 80 taxa of mostly agriculturally important legumes were surveyed as sources of the metabolites, genistein and daidzein. Remarkably high concentrations (over 2 g.kg-1 dry weight) of the anticancer metabolite, genistein, were found in the leaves of Psoralea corylifolia (Indian bread root). All other legumes, with the exception of fermented soybean miso, had genistein levels < 400 mg.kg-1 dry weight. Concentrations of over 1 g.kg-1 dry weight and 0.95 g.kg-1 dry weight of the anticancer metabolite, daidzein, were found in the stems of the fava bean (Vicia faba) and roots of kudzu vine (Pueraria lobata), respectively. From this survey, our results indicate that the legumes, lupine (Lupinus spp.), fava bean, (Vicia faba), soybeans (Glycine max), kudzu (Pueraria lobata), and psoralea (Psoralea corylifolia), are excellent food sources for both genistein and daidzein. Miso, a fermented soybean product, is also a rich source of both isoflavones.

Function of silica bodies in the epidermal system of rice (Oryza sativa L.): testing the window hypothesis.

Silicon has been considered to be important for normal growth and development of the rice plant (Oryza sativa L.). To investigate the physiological function of deposited silica in rice leaves, the hypothesis that silica bodies in the leaf epidermal system might act as a 'window' to facilitate the transmission of light to photosynthetic mesophyll tissue was tested. The silica content of leaves increased with supplied silicon and was closely correlated with the number of silica bodies per unit leaf area in the epidermal system. There was a significant difference in silica deposition and formation of silica bodies between Si-treated and non-treated leaves; silicon was polymerized inside the silica cells and bulliform cells of the epidermis, in Si-treated leaves. Although the 'windows' were only formed in leaves with applied silicon, optical properties of leaf transmittance, reflectance and absorptance spectra in Si-treated and non-treated leaves were almost equal. Furthermore, light energy use efficiency and quantum yield of Si-treated leaves were less than in leaves not containing silica bodies. Thus, silica bodies, at least based on the data, do not function as windows in rice leaves.

Basis for changes in the auxin-sensitivity of Avena sativa (oat) leaf-sheath pulvini during the gravitropic response.

During the gravitropic response, auxin-sensitivity of the lower flanks of leaf-sheath pulvini of Avena sativa (oat) is at least 1000-fold higher than those of the upper flanks and non-gravistimulated pulvini. When the pulvini are treated with 1 mM Ca2+, a 10-fold increase in auxin-sensitivity of the pulvini is observed. Related to this difference in auxin-sensitivity, in vitro activation of the vanadate-sensitive H(-)-ATPase by IAA was observed. Results show that the activation of the H(+)-ATPase by IAA is probably mediated by soluble protein factors and that the H(+)-ATPase prepared from the lower flanks is activated by IAA with a 1000-fold higher auxin-sensitivity as compared with that from the upper flanks of the graviresponding pulvini. Ammonium sulfate fractionation experiments show that these soluble protein factors are in the 30 to 60% fraction. Auxin-binding assays reveal that lower flanks contain more high-affinity soluble auxin-binding sites (kD; on the order of 10(-9) M) and less low-affinity soluble auxin-binding sites (kD; on the order of 10(-6) M) than upper flanks. It is concluded that differential auxin-sensitivity of graviresponding oat-shoot pulvini is achieved by the modulation of affinities of auxin-binding sites in upper and lower flanks of the pulvini, that Ca2+ is involved in such modulation, and that one of the probable cellular functions of these auxin binding sites is the activation of the proton pump on the plasma membranes.

Molecular basis of the increase in invertase activity elicited by gravistimulation of oat-shoot pulvini.

An asymmetric (top vs. bottom) increase in invertase activity is elicited by gravistimulation in oat-shoot pulvini starting within 3 h after treatment. In order to analyze the regulation of invertase gene expression in this system, we examined the effect of gravistimulation on invertase mRNA induction. Total RNA and poly (A)+RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino-acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial-length cDNA (550 base pairs) was obtained and characterized. There was a 52% deduced amino-acid sequence homology to that of carrot beta-fructosidase and a 48% homology to that of tomato invertase. Northern blot analysis showed that there was an obvious transient accumulation of invertase mRNA elicited by gravistimulation of oat pulvini. The mRNA was rapidly induced to a maximum level at 1 h following gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher (five-fold) than that in the top half of the pulvinus tissue. The induction of invertase mRNA was consistent with the transient enhancement of invertase activity during the graviresponse of the pulvinus. These data indicate that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional and/or translational levels. Southern blot analysis showed that there were four genomic DNA fragments hybridized to the invertase cDNA. This suggests that an invertase gene family may exist in oat plants.

Gibberellin (GA3) enhances cell wall invertase activity and mRNA levels in elongating dwarf pea (Pisum sativum) shoots.

The invertase (EC 3.2.1.26) purified from cell walls of dwarf pea stems to homogeneity has a molecular mass of 64 kilodaltons (kD). Poly(A)+RNA was isolated from shoots of dwarf pea plants, and a cDNA library was constructed using lambda gt11 as an expression vector. The expression cDNA library was screened with polyclonal antibodies against pea cell wall invertase. One invertase cDNA clone was characterized as a full-length cDNA with 1,863 base pairs. Compared with other known invertases, one homologous region in the amino acid sequence was found. The conserved motif, Asn-Asp-Pro-Asn-Gly, is located near the N-terminal end of invertase. Northern blot analysis showed that the amount of invertase mRNA (1.86 kb) was rapidly induced to a maximal level 4 h after GA3 treatment, then gradually decreased to the control level. The mRNA level at 4 h in GA3-treated peas was fivefold higher than that of the control group. The maximal increase in activity of pea cell wall invertase elicited by GA3 occcured at 8 h after GA3 treatment. This invertase isoform was shown immunocytochemically to be localized in the cell walls, where a 10-fold higher accumulation occurred in GA3-treated tissue compared with control tissue. This study indicates that the expression of the pea shoot cell-wall invertase gene could be regulated by GA3 at transcriptional and/or translational levels.

Kinetic induction of oat shoot pulvinus invertase mRNA by gravistimulation and partial cDNA cloning by the polymerase chain reaction.

An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)+ RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 h after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

The role of calcium in growth induced by indole-3-acetic acid and gravity in the leaf-sheath pulvinus of oat (Avena sativa).

Leaf-sheath pulvini of excised segments from oat (Avena sativa L.) were induced to grow by treatment with 10 micromoles indole-3-acetic acid (IAA), gravistimulation, or both, and the effects of calcium, EGTA, and calcium channel blockers on growth were evaluated. Unilaterally applied calcium (10 mM CaCl2) significantly inhibited IAA-induced growth in upright pulvini but had no effect on growth induced by either gravity or gravity plus IAA. Calcium alone had no effect on upright pulvini. The calcium chelator EGTA alone (10 mM) stimulated growth in upright pulvini. However, EGTA had no effect on either IAA- or gravity-induced growth but slightly diminished growth in IAA-treated gravistimulated pulvini. The calcium channel blockers lanthanum chloride (25 mM), verapamil (2.5 mM), and nifedipine (2.5 mM) greatly inhibited growth as induced by IAA (> or = 50% inhibition) or IAA plus gravity (20% inhibition) but had no effect on gravistimulated pulvini. Combinations of channel blockers were similar in effect on IAA action as individual blockers. Since neither calcium ions nor EGTA significantly affected the graviresponse of pulvini, we conclude that apoplastic calcium is unimportant in leaf-sheath pulvinus gravitropism. The observation that calcium ions and calcium channel blockers inhibit IAA-induced growth, but have no effect on gravistimulated pulvini, further supports previous observations that gravistimulation alters the responsiveness of pulvini to IAA.

Changes in the ultrastructure of cell walls, cellulose synthesis, and glucan synthase activity from gravistimulated pulvini of oat (Avena sativa).

Ultrastructural analyses of the cell walls from top and bottom halves of gravistimulated pulvini from oat leaves show a decrease in the density of material within the cell walls from the lower halves of pulvini after 24 h of gravistimulation. Assays of cellulose synthesis with a 14C-sucrose pulse-chase experiment indicate no difference in the amount of new cellulose synthesized in top compared with bottom halves of gravistimulated pulvini. The highest rate of cellulose synthesis occurs with 12-24 h of gravistimulation. Treatment of graviresponding pulvini with 2,6-dichlorobenzonitrile (DCBN) had only a minor effect on segment gravitropic curvature. We also found that there is no difference in the activities of either glucan synthase I or glucan synthase II in top halves as compared with bottom halves of gravistimulated pulvini. We conclude that the graviresponse in oat stems is not driven by new cell wall synthesis but, rather, by changes in cell wall plasticity and osmotic potential.

Green and non-green callus induction from excised rice (Oryza sativa) embryos: effects of exogenous plant growth regulators.

Calli were induced either from excised rice embryos or from whole seeds in the presence of 1 to 5 mg l-1 NAA. After 12 days of culture, calli were induced only from excised rice embryos. We found that excised embryos accumulated NAA up to 6 times higher concentration than did whole seeds. In the presence of 1 to 5 mg l-1 NAA and 2 to 10 mg l-1 kinetin, chlorophyllous calli were induced from excised rice embryos. Chlorophyll contents in the callus tissue increased with increasing kinetin concentration while percent callus induction decreased. The total chlorophyll content was linearly correlated with the ratio of kinetin to NAA in the medium.

Dynamics of auxin movement in the gravistimulated leaf-sheath pulvinus of oat (Avena sativa).

The role of auxin redistribution in the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa L.) was assessed using 3H-indole-3-acetic acid (3H-IAA) preloaded into isolated pulvini. When pulvini were totally isolated from subtending nodal tissue as well as leaf-sheath and internode, gravistimulation failed to induce an asymmetric growth response. Presence of either the nodal tissue or the internode/leaf-sheath tissue was sufficient to restore a normal graviresponse. Gravistimulation of totally isolated pulvini inhibited basipetal export of label (i.e., 3H-IAA) without generating any asymmetry of label within the pulvinus. In contrast, gravistimulation of pulvini with nodes intact generated an asymmetric distribution of label (initiation by 1 h; final ratio, lower/upper = 1.5) as well as the upward bending response. The kinetics of formation of the asymmetry of label paralleled the kinetics of initiation of the asymmetric growth response. The addition of 0.1 M sucrose to all agar blocks shortened both the time to initiation of label redistribution and the time to initial upward bending. However, sucrose did not change the final magnitude of label asymmetry although it increased the final steady state bending rate four fold. The inhibitors of polar auxin transport N-1-naphthylphthalamaic acid (NPA), 2,3,5-triiodobenzoic acid (TIBA), morphactin, naringenin, kaempferol and myricetin all significantly decreased the bending response of oat pulvini, but this inhibition was less than 50%. In contrast, TIBA and naringenin (each at 100 micromoles), effectively eliminated the redistribution of label, but did not eliminate the bending response. These results indicate that the active basipetal export of auxin is inhibited by gravistimulation of the oat pulvinus, while active lateral transport is induced. It is concluded that, while lateral transport of auxin occurs following gravistimulation, it is not necessary for a graviresponse. Other processes, such as localized changes in tissue responsiveness or the conversion of conjugated hormone to free (active) hormone, may suffice to drive the graviresponse.

Elongation growth of the leaf sheath base of Avena sativa seedlings: regulation by hormones and sucrose.

The leaf sheath base of the seedling of Avena sativa was characterized for growth response to hormones and sucrose. Six day old plants, raised under a 10:14 hr light:dark cycle, were excised at the coleoptilar node and 1 cm above the node for treatment. The growth of the leaf sheath base was promoted by gibberellic acid (GA3) and this response was dose dependent. The lag to response initiation was approximately 4 hr. Growth with or without GA3 (10 micromoles) was transient, diminishing appreciably after 48 hr. The addition of 10 mM sucrose greatly prolonged growth; the effect of GA3 and sucrose was additive. Neither indole-3-acetic acid (IAA) nor the cytokinin N6-benzyladenine (BA), alone or in combination, promoted the growth of leaf sheath bases. However, both significantly inhibited the action of GA3. The inhibitory effect of IAA was dose dependent and was not affected by the addition of BA or sucrose. These results indicate that the growth of leaf sheath bases of Avena sativa is promoted specifically by gibberellin, that this action depends on the availability of carbohydrates from outside of the leaf sheath base, and that the promotional effect of GA3 can be modified by either auxins or cytokinins.

Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa).

The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response accompanied by several asymmetric processes, including degradation of starch and cell wall synthesis. To understand further the cellular and biochemical events associated with the graviresponse, changes in cell walls and their constituents and the activities of related enzymes were investigated in excised pulvini. Asymmetric increases in dry weight with relatively symmetric increases in wall weight accompanied the graviresponse. Starch degradation could not account for increases in wall weight. However, a strong asymmetry in invertase activity indicated that hydrolysis of exogenous sucrose could contribute significantly to the increases in wall and dry weights. Most cell wall components increased proportionately during the graviresponse. However, beta-D-glucan did not increase symmetrically, but rather increased in proportion in lower halves of gravistimulated pulvini. This change resulted from an increase in glucan synthase activity in lower halves. The asymmetry of beta-D-glucan content arose too slowly to account for initiation of the graviresponse. A similar pattern in change in wall extensibility was also observed. Since beta-D-glucan was the only wall component to change, it is hypothesized that this change is the basis for the change in wall extensibility. Since wall extensibility changed too slowly to account for growth initiation, it is postulated that asymmetric changes in osmotic solutes act as the driving factor for growth promotion in the graviresponse, while wall extensibility acts as a limiting factor during growth.